In the last decade, the administration of therapeutic genes to patients has become a reality for preventing or treating various diseases. Non-viral vectors are often preferred in clinical applications to minimise the risk of viral infections. This increases the demand for highly purified plasmids for use in gene therapy and plasmid-based vaccines. The stringent guidelines and rules set forth by health authorities require homogeneous preparations of purified supercoiled plasmid DNA for clinical applications.
Chromatography is the method of choice for both small- and large-scale purification of supercoiled plasmid DNA (Ferreira, G. N. M., Prazeres, D. M. F., Cabral, J. M. S., and Schleef, M. Plasmid manufacturing—An overview. In: Schleef, M. (Ed.) Plasmids for therapy and vaccination Wiley-VCH: Weinheim, 2001, p. 193-236). Separation methods based on size-exclusion, ion-exchange and hydrophobic interaction chromatography (HIC) have been shown to be suitable for purifying plasmid DNA (Ferreira, G. N. M., Monteiro, G. A., Prazeres, D. M. F., and Cabral, J. M. S. Downstream processing of plasmid DNA for gene therapy and DNA vaccine applications. TIBTECH 2000; 18, 380-388). Affinity chromatography, which is based on sequence-specific interactions between an immobilised synthetic oligonucleotide and a stretch of the plasmid DNA, has also been suggested (Schluep, T., and Cooney, C. L. Purification of plasmids by triplex affinity interaction. Nucleic Acids Research 1998; 26, 4524-4528). However, none of these techniques results in a homogeneous preparation of supercoiled plasmid DNA in sufficient quantities for non-analytical applications.
To circumvent this problem, efforts in the past were directed towards the development of sample preparation techniques that would minimise the formation of nicked forms of plasmid DNA. However, this approach was difficult to reproduce and the quality of the plasmid DNA thus produced did not meet the stringent specifications set forth for the final product (Levy, M. S., O'Kennedy, R. D., Ayazi-Shamlou, P., and Dunnill, P. Biochemical engineering approaches to the challenges of producing pure plasmid DNA. Trends in Biotechnology 2000; 18, 296-305). Accordingly, there has been a recognised need for developing a new and robust separation protocol for supercoiled plasmid DNA that would significantly reduce the tedious efforts for optimisation of the upstream processes.
Oscarsson et al. suggested thiophilic chromatography (Oscarsson, S., and Porath, J. Covalent chromatography and salt-promoted thiophilic adsorption. Analytical Biochemistry 1989; 176, 330-337; Porath, J., Maisano, F., and Belew, M. Thiophilic adsorption—a new method for protein fractionation. FEBS Letters 1985; 185, 306-310) to protein purification. In a patent application of later date, WO 95/33557, Oscarsson and Porath disclose an alkali-resistant protein adsorbent, which is similar to the ones discussed in 1989, but wherein the thiophilic group has been distanced from the rest of the ligand to improve the properties in alkaline environments. Adsorption of proteins is favoured by high concentrations of lyotropic salt, and desorption is achieved using the conditions conventionally used in hydrophobic interaction chromatography (HIC), which is to replace the lyotropic salt by another, less lyotropic salt or simply by water.
More recently, the ability of different ligand structures comprising thioethers to bind the different isoforms of plasmid DNA under conditions that would be applicable in large-scale processes have been investigated. This was disclosed in Swedish patent application SE 0101380.4, which however was not published at the time of the filing of the present application. Accordingly it was possible to narrow down the specific structures that are required for an “optimal ligand” to interact with supercoiled plasmid DNA and purify it selectively. As a result, a new group of thiophilic ligands, namely aromatic thioethers, have been identified, which effectively differentiates between the isoforms of plasmid DNA. Media comprising these ligands have been shown to efficiently separate supercoiled (covalently closed circular (ccc)) plasmid DNA from its isoform, i.e. open circular (oc) form in a single chromatography step. Accordingly, the use of these media appears to be promising and should facilitate the production of highly purified supercoiled plasmid DNA for use in gene therapy and DNA vaccine applications.
However, even though plasmid DNA can be separated from its isoform, other problems remain to be solved. For example, a cell lysate comprising a desired nucleic acid will usually also comprise one or more proteins, such as enzymes, or various degradation products. Even though previously reported methods in principle are capable of separating protein from desired nucleic acids, the resolution obtained is still not fully satisfactory for an efficient production process. Cell lysates that comprise deoxynucleic acid (DNA) will normally also comprise ribonucleic acid (RNA) and some remaining proteins. However, in the above discussed context when plasmid DNA is desired for pharmaceutical purposes, both will constitute contaminants that has to be removed. Furthermore, chromatographic purification of plasmid DNA results in most cases in co-purification of endotoxins, i.e. membrane components of host bacteria, that have to be removed to fulfil the rigorous requirements for pharmaceutical products. Accordingly, there is a need of improvements of the presented purification protocols using thiophilic chromatography as regards the separation of nucleic acids from the above-discussed contaminants.